Recent advances in imaging biological cells using optical tomography have been developed by Nelson as disclosed, for example, in U.S. Pat. No. 6,522,775, issued Feb. 18, 2003, and entitled “APPARATUS AND METHOD FOR IMAGING SMALL OBJECTS IN A FLOW STREAM USING OPTICAL TOMOGRAPHY,” the full disclosure of which is incorporated by reference. Further development in the field is taught in Fauver et al., U.S. patent application Ser. No. 10/716,744, filed Nov. 18, 2003 and published as US Publication No. US-2004-0076319-A1 on Apr. 22, 2004, entitled “METHOD AND APPARATUS OF SHADOWGRAM FORMATION FOR OPTICAL TOMOGRAPHY,” the full disclosure of which is also incorporated by reference.
Processing in such an optical tomography system begins with specimen preparation. Typically, specimens taken from a patient are received from a hospital or clinic and processed to remove non-diagnostic elements, fixed and then stained. Stained specimens are then mixed with an optical gel, inserted into a micro-capillary tube and images of objects, such as cells, in the specimen are produced using an optical tomography system. The resultant images comprise a set of extended depth of field images from differing perspectives called “pseudoprojection images.” The set of pseudoprojection images can be reconstructed using backprojection and filtering techniques to yield a 3D tomogram of a cell of interest.
The 3D tomogram then remains available for analysis in order to enable the quantification and the determination of the location of structures, molecules or molecular probes of interest. An object such as a biological cell may be labeled with at least one stain or tagged molecular probe, and the measured amount and location of this probe may yield important information about the disease state of the cell, including, but not limited to, various cancers such as lung, breast, prostate, cervical and ovarian cancers.
In Optical Tomography Microscope (OPTM) systems as described, for example, in Fauver, about 250 sample images taken over a 180 degree rotation are required to adequately sample the volume of a cell nucleus randomly distributed in a flow stream within a 50 micron capillary tube. Due to limitations in the previous cell introduction method, a high number of the cells appear close to the capillary tube walls making the sampling just good enough to render ˜0.6 micron resolution at an outer radius.
Because such optical tomography systems use unfocused capillary tube loading techniques, cells and other objects are prone to tracking errors and optical imperfections including geometric distortion and loss of resolution from aberrations induced by tube wall refraction. Such systems are also sensitive to longitudinal movement due to vibration of media, temperature changes, entrapped gas expansion and/or gel instability from chemistry and local rheology changes. Uncentered specimens also tend to stick to walls or move slowly along walls leading to clogging from aggregations of cells attaching to walls. Present systems also suffer from sample carryover problems.
In order to improve throughput, a method for providing higher resolution or improved signal to noise is needed to reduce sampling requirements while maintaining acceptable resolution. The present invention provides new and novel techniques for centering samples and reducing sample volumes to improve image acquisition and throughput in an OPTM system while mitigating sample carryover issues.